Eubacteria and archaea use cytoskeletal elements including, actin-like filaments, tubulin- related polymers, and even intermediate filaments, to: (1) control their shape;(2) to divide;(3) to establish order in the cytoplasm;and (4) to move intracellular cargo. A long-term goal of my laboratory is to chart the structural and biochemical diversity of bacterial cytoskeletal proteins (especially actin-like proteins) and to understand how the unique properties of each are adapted to its function. In this project we focus on a gram positive actin-like protein, called AlfA, which stabilizes plasmids in Bacillus subtilis during both vegetative growth and sporulation. We focus on AlfA for three reasons: (1) It provides the first opportunity to study a cargo-hauling actin from a gram positive organism. (2) It is involved in segregating a stable plasmid in a commercially important strain of B. subtilis (natto) and may be related to systems that maintain virulence factors in gram positive pathogens. (3) Preliminary experiments reveal that the structure and assembly dynamics of AlfA are dramatically different from those of any previously characterized actin. Namely, in preliminary experiments we found that AlfA: (i) lacks the dynamic instability which is key to the cellular function of other DNA-segregating polymers and (ii) assembles into two-stranded helical filaments, which spontaneously associate into stable, mixed-polarity bundles. Live-cell imaging suggests that these stable bundles are the functional form of AlfA and reveals that AlfA filaments simultaneously assemble and disassemble (treadmill) inside these bundles. Together these observations rule out the possibility that AlfA segregates DNA by any previously proposed mechanism and suggest that AlfA forms a bi-directional treadmill that continuously carries plasmids to the poles of Bacillus cells (and into the forespore during sporulation). The present proposal is aimed at uncovering the mechanism by which AlfA segregates and stabilizes plasmids and determining how the unique properties of AlfA enable it to carry out this task. PUBLIC HEALTH RELEVANCE: We are just beginning to understand the details of how bacteria control their shapes, organize their insides, and divide. These processes all require the assembly of complex molecular scaffolds, called cytoskeletal networks. Understanding the assembly and function of these networks will enable us to better understand how pathogens acquire and maintain drug resistance (segregation of drug resistance plasmids) and provide new targets for antibiotic therapy (e.g. cytoskeletal proteins that control cell growth and division).